An understanding of the events involved in the regulation of the initiation of transcription in mammalian cells awaits the development of in vitro systems that faithfully reproduce in vivo situations. One requirement for the genesis of such in vitro systems is the ability to generate DNA templates arranged as chromatin. The bovine papilloma virus type I (BPV) is an eukaryotic plasmid vector that can allow the chromatin structure of promoters mobilized on it to be isolated from cultured cells. Therefore, this vector provides a way to establish chromatin structure in vitro. The long term goals of the work proposed here are to use this episomal system to dissect, at the molecular level, the regulation of the mouse mammary tumor virus (MMTV) promoter by glucocorticoid hormones, and to extend the use of BPV vectors to promoters that are regulated by peptide hormones and growth factors, so that this type of cellular gene regulation may become amenable to in vitro manipulation. The specific aims of this proposal are three-fold. First, MMTV-BPV mini-chromosomes will be used to reconstitute steroid regulation in vitro. These studies will provide the assay necessary for defining the molecular events that lead to the regulation of MMTV transcription. Second, proteins uniquely bound to MMTV-BPV episomes will be isolated and characterized, and used as antigen for the production of monoclonal antibodies. These experiments will provide the tools required to fully develop the in vitro assay for hormone modulation of transcription. Third, a promoter regulated by peptide factors will be introduced into cultured cells via BPV vectors, and its ability to be regulated at an extrachromosomal locus will be tested. This work will use the MMTV system as a blueprint for the development of in vitro assays for transcriptional regulation mediated by peptide hormones. This final aim should lay the foundation for understanding how agents that act at the cell surface effect the transcription of specific genes, a concept of special interest in view of the importance of peptide factors in both normal development and malignant transformation of cells.